Methods and reagents for inhibiting furin endoprotease

ABSTRACT

This invention relates to method and reagents for inhibiting furin endoprotease activity and specifically for inhibiting furin endoprotease-mediated maturation of bioactive proteins in vivo and in vitro. The invention specifically provides proteins capable of inhibiting furin endoprotease activity. Particularly provided are α 1  -antitrypsin variants that specifically inhibit furin endoprotease activity. Methods for using furin endoprotease inhibition to attenuate or prevent viral protein maturation, and thereby alleviate viral infections, are provided. Also provided are methods for using furin endoprotease inhibition to attenuate or prevent proteolytic processing of bacterial toxins, thereby alleviating bacterial infections. Methods are also provided to inhibit proteolytic processing of biologically active proteins and peptides. The invention also provides pharmaceutically acceptable compositions of therapeutically effective amounts of furin endoprotease inhibitors.

This invention was made with government support under DK44629 andDK37274 from the National Institutes of Health. The government hascertain rights in the invention.

This application is a continuation-in-part of Ser. No. 08/002,202 fileJan. 8, 1993, now U.S. Pat. No. 5,604,201.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to endoproteases, particularly a novelendoprotease termed furin endoprotease. The invention also relates toinhibitors of furin endoprotease activity. In particular, the inventionrelates to novel variants of α₁ -antitrypsin that specifically inhibitfurin endoprotease activity. The invention also provides methods forusing such inhibitors to attenuate or prevent biological proteolyticmaturation of bioactive proteins and peptides in vivo and in vitro, inparticular viral proteins and bacterial toxins. Therapeutic methods andpharmaceutical compositions of such inhibitors are also provideddirected towards the alleviation and treatment of disease havingmicrobiological etiology.

2. Background of the Related Art

Most biologically active peptides and proteins are synthesized initiallyas larger, inactive precursor proteins that are endoproteolyticallycleaved during transit through the secretory pathway in the Golgiapparatus in cells expression such proteins (see Barr, 1991, Cell 66:1-3 for review). This system comprises an important common mechanismrequired for synthesis of biologically active proteins and peptides inyeast (Fuller et al., 1988, Ann. Rev. Physiol. 50: 345-362),invertebrates (Scheller et al., 1983, Cell 32: 7-22) and mammalian cells(Sossin et al., 1989, Neuron 2: 1407-1417). Examples of proteinsproduced in vivo by exocytotic transport through the Golgi areprecursors of peptide hormones, neuropeptides, growth factors,coagulation factors, serum albumin, cell surface receptors, and adhesionmolecules.

Morrison et al., 1985, J. Virol. 53: 851-857 disclose that F protein ofNewcastle disease virus is processed through the exocytotic transportpathway in infected cells.

Perez & Hunter, 1987, J. Virol. 61: 1609-1614 disclose that the Roussarcoma virus (RSV) glycoprotein is processed through the exocytotictransport pathway in infected cells.

Yamada et al., 1988, Virology 165: 268-273 disclose that F protein ofmumps virus is processed through the exocytotic transport pathway ininfected cells.

Randolph et al., 1990, Virology 174: 450-458 disclose that the prMprotein of flaviviruses is processed through the exocytotic transportpathway in infected cells.

A common structural feature of molecules processed through theexocytotic transport pathway is the presence of basic residues or pairsof basic residues at the proteolytic processing site in the molecule.Examples include serum factors (Factor IX; Bentley et al., 1987, Cell45: 343-348; proalbumin; Knowles et al., 1980, Science 209: 497-499;pro-von Willibrand factor; Bonthron et al., 1986, Nature 324: 270-273),viral polyproteins (human immunodeficiency virus (HIV) gp160; McCune etal., 1988, Cell 53: 55-67; RSV envelope protein; Perez & Hunter, 1987,J. Virol. 61: 1609-1614; yellow fever virus protein; Rice et al., 1985,Science 229: 726-733; measles virus protein; Richardson et al., 1986,Virology 155: 508-523; mumps virus protein; Waxham et al., 1987,Virology 159: 381-389; human cytomegalovirus protein; Spaete et al.,1990, J. Virol. 64: 2922-2931; varicella zooster virus protein; Kelleret al., 1986, Virology 152: 181-191), growth factors(pre-protransforming growth factor β; Gentry et al., 1988, Molec. Cell.Biol. 8: 4162-4168; epidermal growth factor; Gray et al., 1983, Nature303: 722-725; pro-β-nerve growth factor (NGF); Edwards et al., 1988,Molec. Cell Biol. 8: 2456-2464), receptors (insulin receptor; Yoshimasaet al., 1988, Science 240: 784-787); and bacterial toxins (see Stephen &Pietrowski, 1986, Bacterial Toxins, 2d ed. (Amer. Soc. Microbiol.Washington, D.C.) for review; anthrax toxin; Singh et al., 1989, J.Biol. Chem. 264: 11099-11102). The proteolytic processing site has beenidentified in some of these molecules.

Berger & Shooter, 1977, Proc. Natl. Acad. Sci. USA 74: 3647-3651disclose the sequence -RSKR- (SEQ ID NO.: 1) at the proteolyticprocessing site of pro-β-NGF.

Bentley et al., 1986, ibid. disclose the sequence -RPKR- (SEQ ID NO.: 2)at the proteolytic processing site of the blood coagulation factorprotein Factor IX.

McCune et al., 1988, ibid., disclose the sequence -REKR- (SEQ ID NO.: 3)at the proteolytic processing site of HIV gp160.

Clepak et al., 1988, Biochem. Biophys. Res. Comm. 157: 747-754 disclosethe sequence -RVRR- (SEQ ID NO.: 4) at the proteolytic processing siteof diphtheria toxin.

Vey et al., 1992, Virology 188: 408-413 disclose the sequence -RX(R/K)R-(SEQ ID NO.: 5) at the proteolytic processing site of influenzahemagglutinin.

Ogata et al., 1990, J. Biol. Chem. 265: 20678-20685 disclose thesequence -RSKR- (SEQ ID NO.: 1) at the proteolytic processing site ofPseudomonas exotoxin A.

Klimpel et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10277-10281disclose the sequence -RX(R/K)R- (SEQ ID NO.: 5) at the proteolyticprocessing site of anthrax protective antigen.

Recently, an endoprotease termed furin has been isolated thatspecifically recognizes the recognition sequence of proteins processedthrough the exocytotic secretory pathway (Wise et al., 1990, Proc. Natl.Acad. Sci. USA 87: 9378-9382; Bresnahan et al., 1990, J. Cell Biol. 111:2851-2859). This endoprotease is a subtilisin-related, calcium-dependentserine protein (Bresnahan et al., ibid.). A complementary DNA copy ofthe mRNA encoding this endoprotease has been isolated (Wise et al.,ibid.) and sequenced (van den Ouweland et al., 1992, Nucleic Acids Res.18: 664) and expressed in heterologous cells (Bresnahan et al., ibid.).These studies have shown furin to be expressed as a doublet of 96 and 90kilodaltons (kD) in size, ubiquitously expressed as a 4.5 kilobase (kb)mRNA, and localized by fluorescence immunohistochemistry to the Golgiapparatus of cells expressing this endoprotease (Bresnahan et al.,ibid.). Furin has been shown to be capable of proteolytically cleaving anumber of exocytotically processed proteins.

Bresnahan et al., ibid., disclose furin-mediated cleavage of pro-β-NGF.

Wise et al., ibid., disclose furin-mediated cleavage of pro-vonWillibrand factor and complement factor C3.

Hosaka et al., 1991, J. Biol. Chem. 266: 12127-12130 disclosefurin-mediated cleavage of renin.

Steineke-Grober et al., 1992, EMBO J. 11: 2407-2414 disclosefurin-mediated cleavage of influenza hemagglutinin.

Klimpel et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10277-10281disclose furin-mediated cleavage of anthrax protective antigen.

Molloy et al., 1992, J. Biol. Chem 267: 16396-16402 disclosefurin-mediated cleavage of anthrax protective antigen.

Klimpel et al., 1992, Annual Meeting, Amer. Soc. Microbiol. Abst. B-32disclose furin-mediated cleavage of diphtheria toxin.

Furin can be inhibited by specific peptidyl chloroalkylketones (Gartenet al., 1989, Virology 172: 25-31; Molloy et al., ibid.; Hallenberger etal., 1992, Nature 360: 358-361), but these substances are toxic in vivo.Given the importance of the endoprotease in activation of bacterialtoxins, viral structural proteins and bioactive molecules, there is aneed for the development of safe and specific furin inhibitors.

SUMMARY OF THE INVENTION

This invention provides safe, specific and effective inhibitors of furinendoprotease that are novel variants of the naturally-occurring proteaseinhibitor, α₁ -antitrypsin (SEQ ID NO.: 6) (Heeb et al., 1990, J. Biol.Chem 265: 2365-2369; Schapira et al., 1987, J. Clin. Invest. 80:582-585) and peptides derived therefrom. Use of these novel variants ofα₁ -antitrypsin is advantageous because α₁ -antitrypsin and variants aresecreted proteins that are processed by the exocytotic secretory pathwaythrough the Golgi, so synthesis of these proteins in a cell would resultin delivery of the inhibitor to the site of furin activity in vivo.

In a first embodiment, the invention provides a furin endoproteaseinhibitor comprising an α₁ -antitrypsin variant protein having an aminoacid sequence comprising the amino acids of the native α₁ -antitrypsinmolecule (disclosed in Long et al., 1984, Biochemistry 23: 4828-4837,incorporated by reference), except that the sequence at position 355-358of the native protein (-Ala-Ile-Pro-Met-) (SEQ ID NO.: 7) is changed tothe novel sequence -Arg-Xaa-Xaa-Arg- (SEQ ID NO.: 8), wherein each Xaais any amino acid, at positions 355-358 of the native α₁ -antitrypsinamino acid sequence. In a preferred embodiment, the α₁ -antitrypsinvariant is α₁ -antitrypsin Portland (SEQ ID NO.: 9) and the amino acidsequence at positions 355-358 of the α₁ -antitrypsin amino acid Portlandand the amino acid sequence is -Arg-Ile-Pro-Arg- (SEQ ID NO.: 10).

In a second embodiment, the invention provides a nucleic acid having anucleotide sequence that encodes an α₁ -antitrypsin variant proteinhaving an amino acid sequence comprising the amino acids of the nativeα₁ -antitrypsin molecule, except that the sequence at position 355-358of the native protein (-Ala-Ile-Pro-Met-) (SEQ ID NO.: 7) is changed tothe novel sequence -Arg-Xaa-Xaa-Arg- (SEQ ID NO.: 8), wherein each Xaais any amino acid, at positions 355-358 in the variant α₁ -antitrypsinamino acid sequence. In a preferred embodiment, the α₁ -antitrypsinvariant is α₁ -antitrypsin Portland and the amino acid sequence is-Arg-Ile-Pro-Arg- (SEQ ID NO.: 10).

In a third embodiment, the invention provides a recombinant expressionconstruct comprising a nucleic acid having a nucleotide sequenceencoding an α₁ -antitrypsin variant protein with an amino acid sequencecomprising the amino acids of the native α₁ -antitrypsin molecule,except that the sequence at position 355-358 of the native protein(-Ala-Ile-Pro-Met-) (SEQ ID NO.: 7) is changed to the novel sequence-Arg-Xaa-Xaa-Arg- (SEQ ID NO.: 8), wherein each Xaa is any amino acid,at positions 355-358 of the variant α₁ -antitrypsin amino acid sequence.In a preferred embodiment, the α₁ -antitrypsin variant is α₁-antitrypsin Portland and the amino acid sequence is -Arg-Ile-Pro-Arg-(SEQ ID NO.: 10). The recombinant expression construct provided by theinvention is capable of expression α₁ -antitrypsin variant proteins ofthe invention in a culture of transformed cells. In a preferredembodiment, the recombinant expression construct comprises a vacciniavirus-based construct. In a more preferred embodiment, the recombinantexpression construct comprises a recombinant vaccinia virus vectorcovalently linked to the nucleic acid encoding the α₁ -antitrypsinvariant, preferably α₁ -antitrypsin Portland.

The invention also provides a cell culture transformed with therecombinant expression construct encoding an α₁ -antitrypsin variantcapable of expressing the α₁ -antitrypsin variant. In a preferredembodiment, the α₁ -antitrypsin variant is α₁ -antitrypsin Portland.Preferred embodiments of such cell cultures of bacterial cells, yeastcells, insect cells or mammalian cells.

In another embodiment, the invention provides a homogenous compositionof matter comprising an α₁ -antitrypsin variant produced by the cellculture according to the teachings of the invention. In a preferredembodiment, the α₁ -antitrypsin variant is α₁ -antitrypsin Portland.

The invention also provides a furin endoprotease inhibitor comprising α₁-antitrypsin variants capable of blocking endoproteolytic activation ofbacterial toxins. In a preferred embodiment, the α₁ -antitrypsin variantis α₁ -antitrypsin Portland. Pharmaceutically acceptable compositions ofthe α₁ -antitrypsin variants of the invention are also providedcomprising a therapeutically effective amount of α₁ -antitrypsin variantand a pharmaceutically acceptable carrier or diluent.

Also provided by the invention are peptides having an amino acidsequence of about 4 to about 100 amino acids in length, comprising theamino acid sequence -Arg-Xaa-Xaa-Arg-, wherein each Xaa is any aminoacid. In a preferred embodiment, the amino acid sequence is-Arg-Ile-Pro-Arg-.

The invention provides a method of inhibiting bacterial infection ofhuman cells comprising contacting such cells with an α₁ -antitrypsinvariant of the invention. In a preferred embodiment, the α₁ -antitrypsinvariant is α₁ -antitrypsin Portland. In a preferred embodiment, thebacterial infection is caused by Corynebacterium diptheriae. In anotherpreferred embodiment, the bacterial infection is caused by Bacillusanthracis. In yet another preferred embodiment, the bacterial infectionis caused by Pseudomonas aeruginosa.

The invention also provides a method of inhibiting bacterial infectionin a human comprising administering a therapeutically effective amountof an α₁ -antitrypsin variant of the invention in a pharmaceuticallyacceptable carrier. In a preferred embodiment, the α₁ -antitrypsinvariant is α₁ -antitrypsin Portland. In a preferred embodiment, thebacterial infection is caused by Corynebacterium diptheriae. In anotherpreferred embodiment, the bacterial infection is caused by Bacillusanthracis. In yet another preferred embodiment, the bacterial infectionis caused by Pseudomonas aeruginosa.

The invention provides a method of treating humans with a bacterialinfection comprising administering a therapeutically effective amount ofan α₁ -antitrypsin variant of the invention in a pharmaceuticallyacceptable carrier. In a preferred embodiment, the α₁ -antitrypsinvariant is α₁ -antitrypsin Portland. In a preferred embodiment, thebacterial infection is caused by Corynebacterium diptheriae. In anotherpreferred embodiment, the bacterial infection is caused by Bacillusanthracis. In yet another preferred embodiment, the bacterial infectionis caused by Pseudomonas aeruginosa.

Another method provided by the invention for treating humans with abacterial infection comprises administering a combination oftherapeutically effective amount of an α₁ -antitrypsin variant and atherapeutically effective amount of a second antibacterial compound in apharmaceutically acceptable carrier. In a preferred embodiment, the α₁-antitrypsin variant is α₁ -antitrypsin Portland. In a preferredembodiment, the bacterial infection is caused by Corynebacteriumdiptheriae. In another preferred embodiment, the bacterial infection iscaused by Bacillus anthracis. In yet another preferred embodiment, thebacterial infection is caused by Pseudomonas aeruginosa.

Pharmaceutically acceptable compositions effective according to themethods of the invention, comprising a therapeutically effective amountof a furin endoprotease inhibitor capable of blocking endoproteolyticactivation of bacterial toxins and a pharmaceutically acceptable carrieror diluent, are also provided.

The invention provides a method of inhibiting viral infection of humancells comprising contacting such cells with an α₁ -antitrypsin variantaccording to the invention. In a preferred embodiment, the inventionprovides a gene therapy delivery system for a nucleic acid encoding anα₁ -antitrypsin variant comprising the recombinant expression constructof the invention and genetic means for delivery and expression of therecombinant expression construct into the cells of an animal. Apreferred α₁ -antitrypsin variant is α₁ -antitrypsin Portland.Pharmaceutically acceptable compositions comprising a therapeuticallyeffective amount of the gene therapy delivery system and apharmaceutically acceptable carrier or diluent are provided by theinvention. In a preferred embodiment, the viral infection is caused byHuman Immunodeficiency Virus 1 (HIV-1). In another preferred embodiment,the human cells are hematopoietic cells, most preferably T lymphocytes.Other preferred embodiments of viral infections include infection byinfluenza virus.

The invention also provides a method for inhibiting a viral infection inan animal, most preferably a human, comprising administering atherapeutically effective amount of the gene therapy delivery system ofthe invention in a pharmaceutically acceptable carrier. In a preferredembodiment, the virus is Human Immunodeficiency Virus 1 (HIV-1). Inanother preferred embodiment, the virus is cytomegalovirus.

The invention provides a method of treating humans infected with a viruscomprising administering a therapeutically effective amount of the genetherapy delivery system of the invention in a pharmaceuticallyacceptable carrier. In a preferred embodiment, the virus is HumanImmunodeficiency Virus 1 (HIV-1). In another preferred embodiment, thevirus is influenza virus.

The invention provides a method of treating humans infected with a viruscomprising administering a combination of a therapeutically effectiveamount of the gene therapy delivery system of the invention and atherapeutically effective amount of a second antiviral compound in apharmaceutically acceptable carrier. In a preferred embodiment, thevirus is Human Immunodeficiency Virus 1 (HIV-1) and the second antiviralcompound is azidothymidine. In another preferred embodiment, the virusis influenza virus.

The invention also provides a method for treating virus-associatedimmunosuppression in a human comprising administering a therapeuticallyeffective amount of the gene therapy delivery system of the invention ina pharmaceutically acceptable carrier. In a preferred embodiment, thevirus is Human Immunodeficiency Virus 1(HIV-1).

Pharmaceutically acceptable compositions effective according to themethods of the invention, comprising a therapeutically effective amountof the gene therapy delivery system encoding α₁ -antitrypsin variantshaving antiviral properties and a pharmaceutically acceptable carrier ordiluent, are also provided. In a preferred embodiment, the α₁-antitrypsin variant is α₁ -antitrypsin Portland.

The invention also provides a method of inhibiting proteolyticprocessing of a biologically active protein or peptide in a cellcomprising contacting such cells with the gene therapy delivery systemof the invention. Preferred biologically active proteins are pro-β-nervegrowth factor, blood coagulation factor protein Factor IX, pro-vonWillibrand factor, complement factor C3 and renin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates production of native α₁ -antitrypsin (Lane 1), α₁-antitrypsin Pittsburgh (Lane 2) and α₁ -antitrypsin Portland (Lane 3)by BSC-40 cells infected with vaccinia virus recombinant constructs.

FIG. 2 shows inhibition of thrombin and furin by native α₁ -antitrypsin(striped bars), α₁ -antitrypsin Pittsburgh (dotted bars) and α₁-antitrypsin Portland (stipled bars) in vitro.

FIG. 3 is an SDS-PAGE analysis of inhibition showing proteolyticprocessing of pro-β-NGF by α₁ -antitrypsin and variants.

FIG. 4 depicts Western blot analysis showing inhibition of proteolyticprocessing of HIV gp160 by α₁ -antitrypsin and variants.

FIG. 5 illustrates the experimental protocol for assaying thegp41-mediated fusogenic capacity of gp160-producing IP/IRerythroleukemia cells to form syncytia with CD4⁺ human HeLa cells in thepresence and absence of co-expressed α₁ -antitrypsin and variants.

FIGS. 6A through 6F illustrates the results of the syncytium experimentsdescribed in Example 5.

FIGS. 7A and 7B show an autoradiograph of α₁ -antitrypsinPortland-mediated inhibition of pro-β-NGF proteolytic processing inVV:mNGF-infected BSC-40 cells (FIG. 7A) and quantitation of theseresults as a percentage of the autoradiographic density of the processedband (β-NGF) relative to the total autoradiographic density (the sum ofthe β-NGF band plus the pro-β-NGF band).

FIG. 8 depicts the structure of recombinant expression constructspRep4ΔL/Rpα₁ -PIT and pRep4ΔL/Rpα₁ -PDX.

FIGS. 9A through 9C illustrates HIV-infected cultures of HeLa CD4⁺ clone1022 cells (FIG. 9B) and two independent transfectants, PDX-4 (FIG. 9C)and PDX-6 (FIG. 9A) four days post-infection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The production of proteins such as the α₁ -antitrypsin Portland fromcloned genes by genetic engineering is well known. See, e.g., U.S. Pat.No. 4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65. (Thedisclosure of all U.S. patent references cited herein is to beincorporated herein by reference.) The discussion which follows isaccordingly intended as an overview of this field, and is not intendedto reflect the full state of the art. For the purposes of thisdiscussion, the α₁ -antitrypsin variant α₁ -antitrypsin Portland will beused as an exemplar; it will be understood that the discussion appliesto and the invention encompasses all the α₁ -antitrypsin variants of theinvention.

DNA which encodes α₁ -antitrypsin Portland may be obtained, in view ofthe instant disclosure, by chemical synthesis, or by in vitromutagenesis as described in Example 2 herein of the native α₁-antitrypsin DNA sequence. Such native α₁ -antitrypsin DNA can beobtained by screening reverse transcripts of mRNA from appropriate cellsor cultured cell lines, by screening genomic libraries from appropriatecells, or by combinations of these procedures. Screening of mRNA orgenomic DNA may be carried out with probes generated from the known α₁-antitrypsin gene sequence. Probes may be labeled with a detectablegroup such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with know procedures and used inconventional hybridization assays (see Sambrook et al., 1990, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,N.Y.). In the alternative, α₁ -antitrypsin gene sequences may beobtained for in vitro mutagenesis by use of the polymerase chainreaction (PCR) procedure, with the PCR oligonucleotide primers beingproduced from the native α₁ -antitrypsin gene sequence. See U.S. Pat.Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis.

α₁ -antitrypsin variants such as α₁ -antitrypsin Portland may besynthesized in host cells transformed with a recombinant expressionconstruct comprising a DNA sequence encoding an α₁ -antitrypsin variant.Such a recombinant expression construct can also be comprised of avector that is a replicable DNA construct. Vectors are used hereineither to amplify DNA encoding α₁ -antitrypsin Portland and/or toexpress DNA encoding α₁ -antitrypsin Portland. For the purposes of thisinvention, a recombinant expression construct is a replicable DNAconstruct in which a DNA sequence encoding an α₁ -antitrypsin variant isoperably linked to suitable control sequences capable of effecting theexpression of the α₁ -antitrypsin variant in a suitable host. The needfor such control sequences will vary depending upon the host selectedand the transformation method chosen. Generally, control sequencesinclude a transcriptional promoter, an optional operator sequence (inbacteria) or enhancer sequence (in eukaryotic cells) to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

DNA regions are operably linked when they are functionally related toeach other. For example: a promoter is operably linked to a codingsequence if it controls the transcription of the sequence; a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to permit translation. Generally, operably linked means contiguousand, in the case of leaders sequences, contiguous and in the sametranslational reading frame.

Vectors useful for practicing the present invention include plasmids,viruses (including phage), retroviruses, and integratable DNA fragments(i.e., fragments integratable into the host genome by homologousrecombination). The vector may replicate and function independently ofthe host genome, or may, in some instances, integrate into the genomeitself. Suitable vectors will contain replicon and control sequenceswhich are derived from species compatible with the intended expressionhost. A preferred vector is the plasmid pZVneo, useful for producingrecombinant expression constructs based on homologous recombination withvaccinia virus sequences.

A preferred embodiment of the recombinant expression constructs of thisinvention comprise vaccinia virus sequences capable of infectingmammalian cells and an expressing α₁ -antitrypsin variant, as describedbelow in Example 2.

Transformed host cells are cells which have been transformed ortransfected with a recombinant expression construct made usingrecombinant DNA techniques and comprising sequences encoding an α₁-antitrypsin variant. Transformed host cells may express α₁ -antitrypsinPortland, but host cells transformed for purposes of cloning oramplifying DNA need not express these sequences.

Cultures of cells, including cells derived from multicellular organisms,are desirable hosts for recombinant α₁ -antitrypsin Portland synthesis.In principal, any cell culture is useful that is capable of beingtransformed with an appropriate recombinant expression construct andexpression α₁ -antitrypsin protein. The invention is preferablypracticed with bacterial, yeast, insect or mammalian cells, however,mammalian cells are more preferred, as illustrated in the Examples.Propagation of bacteria and yeast is well known in the art, andpropagation of mammalian cells in cell culture has become a routineprocedure. See Tissue Culture, Academic Press, Kruse & Patterson,editors (1973). Most preferred mammalian cells are BSC-40 African greenmonkey kidney cells, but other cells, such as human 293 cells, VERO andHeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK,COS-7, CV, and MDCK cells, are also useful.

The invention provides homogeneous compositions of α₁ -antitrypsinPortland produced by transformed cells as provided herein. Suchhomogeneous compositions are intended to be comprised of mammalian α₁-antitrypsin Portland protein that comprises at least 90% of the proteinin such homogenous composition.

The recombinant expression constructs of the present invention areuseful in molecular biology to transform cells in vivo in an animal as amethod for protecting the animal from viral or other infection in cellsthat express furin or a furin-like endoprotease activity that can beinhibited by α₁ -antitrypsin Portland. The invention provides a genetherapy delivery system comprising the recombinant expression constructsof the invention in a configuration that enables safe and efficientintroduction of these sequences into appropriate cells and expression ofα₁ -antitrypsin Portland. For such purposes, retroviral vectors asdescribed in U.S. Pat. No. 4,650,764 to Temin & Watanabe or U.S. Pat.No. 4,861,719 to Miller may be employed, for example. The recombinantexpression constructs of the invention may also be used in gene therapycarried out using homologous recombination. See generally Thomas &Capecchi, 1987, Cell 51: 503-512; Bertling, 1987, Bioscience Reports 7:107-112; Smithies et al., 1985, Nature 317: 230-234. Additionally,alteration of endogenous α₁ -antitrypsin sequences to produce α₁-antitrypsin Portland in cells carrying such altered α₁ -antitrypsinsequences can also be achieved using homologous recombination or othertechniques. Transgenic animals, the tissues of which express the α₁-antitrypsin Portland variant, are also envisioned as additional objectsof this invention.

The peptides of this invention may be generated and/or isolated by anymeans known in the art. It is within the skill of those of ordinaryskill in the art to isolate or chemically synthesize a nucleic acidencoding each of the peptides of the invention. Such nucleic acids areadvantageously utilized as components of recombinant expressionconstructs, wherein the nucleic acids are operably linked withtranscriptional and/or translational control elements, whereby therecombinant expression constructs of the invention are capable ofexpressing the peptides of the invention in cultures of cells,preferably eukaryotic cells, most preferably mammalian cells,transformed with such recombinant expression constructs.

The peptides of the invention may be advantageously synthesized by anyof the chemical synthesis techniques known in the art, particularlysolid-phase synthesis techniques, for example, usingcommercially-available automated peptide synthesizers. Such peptides maybe provided as linear peptides encompassing the amino acid sequence-Arg-Xaa-Xaa-Arg-, where each Xaa is any amino acid. These peptides mayalso be provided in the form of combination peptides, wherein thepeptides comprising the combination are linked in a linear fashion oneto another, with or without separation by "spacer" amino acids allowingfor selected conformational presentation. Also provided arebranched-chain combinations, wherein the component peptides arecovalently linked via functionalities in amino acid sidechains of theamino acids comprising the peptides.

The invention also provides antibacterial and antiviral methods. Theinvention provides methods for blocking endoproteolytic activation ofbacterial toxins. Bacterial targets of the antibacterial methodsprovided by this invention include but are not limited to any bacteriathat produces an endoproteolytically-activated toxin, such as diptheriatoxin produced by Corynebacterium diptheriae, exotoxin A of Pseudomonasaeruginosa, tetanus toxin, the enterotoxins of Escherichia coli andVibrio cholerae, protective antigen of Bacillus anthracis and theneurotoxin and C2 toxin of Clostridium botulinum. Preferred toxins arethose that are proteolytically processed at a consensus furinrecognition site (-Arg-Xaa-Xaa-Arg↓-) (SEQ ID NO.: 8). Preferredembodiments include Corynebacterium diptheriae, Pseudomonas aeruginosaand Bacillus anthracis.

Viral targets of antiviral methods provided include but are not limitedto picornaviruses (e.g., poliovirus and rhinovirus); orthomyxoviruses(e.g., influenza virus); paramyxoviruses (e.g., measles virus and mumpsvirus); coronaviruses; rhabdoviruses (e.g., rabies virus and vesicularstomatitis virus); togaviruses (e.g., Semliki Forest virus and yellowfever virus); bunyaviruses (e.g., California encephalitis virus);arenaviruses (e.g., Lassa fever virus); rubella virus; reoviruses (e.g.,Colorado tick virus); hepatitis viruses; adenoviruses; herpesviruses(e.g., herpes simplex virus); and oncogenic viruses, including papillomaviruses, RNA tumor viruses, or retroviruses, and lentiviruses (e.g.,human immune deficiency virus). The most preferred viruses are the humanimmunodeficiency viruses (HIV-1 and HIV-2).

Cells intended to be protected by the methods provided by this inventioninclude but are not limited to human, canine, bovine, murine, leoporine,porcine, ovine, simian, feline, hircine, and equine cells. The preferredcells are human cells. More preferred cells are human T lymphocytes (Tcells), and the most preferred human T cells are those human T cellsexpressing the cell surface antigen CD4.

The methods of the present invention may be used to treat donated humanblood or plasma to protect transfusion recipients from viral infectionfrom contaminating virus. The methods of the present invention may beused to treat human semen to protect embryos derived from such semen,and mothers bearing such embryos or impregnated with such semen, fromcontaminating virus. In a preferred embodiment, the contaminating virusis HIV-1.

The present invention provides methods for inhibiting viral infection ina human. The invention also provides for treating a human infected witha virus. Another embodiment of the present invention also includesmethods for treating immunosuppression in a human associated with viralinfection. Yet another embodiment of the present invention provides amethod of prophylaxis for treating a human exposed to infection with avirus, in particular those directly at risk of infection as a result ofintimate contact with humans infected with a virus of tissues or bodilyfluids contaminated by a virus. The preferred virus of these embodimentsof the invention is HIV-1. The invention provides pharmaceuticallyacceptable compositions effective for use with the methods provided bythe invention comprising the peptides of the invention and apharmaceutically acceptable carrier.

The invention also provides methods for inhibiting proteolyticprocessing of a biologically active protein or peptide in a cellcomprising contacting such cells with the gene therapy delivery systemof the invention. The methods of the invention encompass inhibition ofproteolytic processing of any biologically active molecule that isproteolytically processed by furin in vivo or in vitro, including butnot limited to peptide hormones, neuropeptides, growth factors,coagulation factors, serum albumin, cell surface receptors, and adhesionmolecules. Preferred biologically active proteins are pro-β-nerve growthfactor, blood coagulation factor protein Factor IX, pro-von Willibrandfactor, complement factor C3 and renin, for alleviation of pathologicalconditions and disease states in an animal, preferably a human,associated with over-expression, over-production or otherwiseinappropriate synthesis of such biologically-active proteins.

Preparation of pharmaceutically acceptable compositions provided by thepresent invention can be prepared using methods well known to those withskill in the art. Any of the common carriers such as sterile salinesolution, plasma, etc., can be utilized for preparing the pharmaceuticalcompositions provided by the invention. Routes of administration includebut are not limited to oral (including inhalation into the lungs),intravenous, parenteral, rectal, optical, aural and transdermal. Thepharmaceutical compositions of the invention may be administeredintravenously in any conventional medium for intravenous injection suchas an aqueous saline medium, or in blood plasma medium. Such medium mayalso contain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. Among the preferred mediaare normal saline and plasma.

The following Examples illustrate certain aspects of the above-describedmethod and advantageous results (also disclosed in Anderson et al.,1993, J. Biol. Chem. 268: 24887-24891). The following Examples are shownby way of illustration and not by way of limitation.

EXAMPLE 1 Production of Furin Endoprotease in African Green Monkey CellsIn Vitro

Human furin was synthesized for inhibition experiments as described inBresnahan et al., (1990, J. Cell Biol. 111: 2851-2859) and Molloy etal., (1992, J. Biol. Chem. 267:16396-14402; both hereby incorporated byreference). Briefly, a furin cDNA (van den Ouweland et al., 1992,Nucleic Acids Res. 18: 664) encoding a truncated but functional furinprotein was inserted into the multiple cloning site of a vaccinia virusvector (see Hruby et al., 1986, Meth. Enzymol. 124: 295-309) and used toinfect BSC-40 African green monkey kidney cells (ATCC Accession No. CCL26, American Type Culture Collection, Rockville, Md.). The cells wereincubated in serum-free defined media for 24 hours and then harvested at4° C. and disrupted by 20-30 strokes in a Dounde homogenizer (KontesGlass Co., Vineland, Ohio). The lysate was cleared by low-speedcentrifugation (1000 g, 5 min) and the supernatant then subjected toultracentrifugation at 100,000 g for 1 hr. The pellet was resuspended in200 μL of a buffer comprising 10 mM HEPES(N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), Sigma ChemicalCo., St. Louis, Mo.), pH 7.5 and stored at 4° C. until use.

Alternatively, a soluble, truncated derivative of furin lacking thecarboxyl-terminal 81 amino acids was used (see Molloy et al., ibid.,incorporated by reference, for a more detailed description of thismolecule). Briefly, BSC-40 cells were infected with a vaccinia virusvector containing the truncated furin cDNA, grown for 16-18 h inserum-free defined media and then harvested. The cells were disruptedand subjected sequentially to centrifugation at 1000 g and 10,000 g at4° C. The supernatant was passed through a 0.2 μm filter and thendiluted approximately 1:1 with a buffer consisting of 20 mM BisTris (pH7.0) and 1 mM 2-mercaptoethanol. This mixture was then applied to a MonoQ HR 5/5 fast protein liquid chromatography anion exchange column(Pharmacia LKB Biotechnology Inc.) and the truncated furin derivativeeluted with a gradient of 0-750 mM sodium chloride and stored at 4° C.until use.

EXAMPLE 2 In Vitro Mutagenesis and Production of Native and VariantSpecies of α₁ -Antitrypsin

A novel α₁ -antitrypsin variant, termed α₁ -antitrypsin Portland (SEQ IDNO.: 9), was produced by in vitro mutagenesis of a cloned cDNA encodingthe naturally-occurring variant α₁ -antitrypsin Pittsburgh (SEQ ID NO.:12-13) (Lewis et al., 1978, Blood 51: 129-137; Owen et al., 1983, N.Engl. J. Med. 309: 694-698). A full length cDNA encoding α₁ -antitrypsinPittsburgh (which is identical to the sequence disclosed in Long et al.,supra, except for a Met₃₅₈ →Arg₃₅₈ mutation) was subcloned into M13mp19phage using standard techniques (see Sambrook et al., ibid., Chapter15). The following mutagenesis primer was then annealed to the sequenceof single-stranded phage DNA corresponding to codons 352-358 of the α₁-antitrypsin Pittsburgh sequence:

    5'-TTTTTAGAGCGCATACCCAG-3'                                 (SEQ ID NO.: 14)

The underlined sequence encodes the mutagenized codon. After annealing,the mutagenesis primer was extended using the Klenow fragment of E. coliDNA polymerase in the presence of the four deoxynucleotidetriphosphates. Clones having the mutagenized sequence were then grownand selected in E. coli, and DNA sequencing of the appropriate portionof the sequence of selected mutagenized clones was performed to confirmsuccessful mutagenesis. The mutagenized α₁ -antitrypsin cDNA sequenceswere then subcloned into the vaccinia virus recombination vector pZVneoand used to produce vaccinia virus recombinants as described in Example1 (and described in more detail in Hayflick et al., 1992, J. Neurosci.12: 705-717, hereby incorporated by reference).

As a result of mutagenesis, the sequence of α₁ -antitrypsin Pittsburgh(Ala₃₅₅ -Ile-Pro-Arg₃₅₈) (SEQ ID NO.: 15) was changed to the novelsequence of α₁ -antitrypsin Portland (Arg₃₅₅ -Ile-Pro-Arg₃₅₈) (SEQ IDNO.: 10). Vaccinia virus constructs containing native α₁ -antitrypsin(SEQ ID NOS.: 16 & 17) (VV:α₁ -NAT; Ala₃₃₅ -Ile-Pro-Met₃₅₈) (SEQ ID NO.:7), α₁ -antitrypsin Pittsburgh (SEQ ID NOS.: 12 & 13), (VV:α₁ -PIT) andα₁ -antitrypsin Portland (SEQ ID NOS.: 18 & 19) (VV:α₁ -PDX) were eachused to infect BSC-40 cells. Such infected cells secrete each of the α₁-antitrypsins into the culture media, and native α₁ -antitrypsin andvariants were isolated from culture media from appropriately-infectedBSC-40 cells by passage of such media over a Mono Q HR 5/5 high pressureliquid chromatography anion exchange column (Pharmacia LKB BiotechnologyLtd., Stockholm, Sweden) and eluted using a linear gradient (0.05→0.5M)of sodium chloride in 50 mM Tris-HCl (pH 8.0), as described further inMolloy et al. Production of native α₁ -antitrypsin (Lane 1) and variantsPittsburgh (Lane 2) and Portland (Lane 3) was confirmed by Western blothybridization (see Sambrook et al., ibid., Chapter 18 ) as shown in FIG.1.

EXAMPLE 3 In Vitro Characterization of Furin Endoprotease Inhibition byα₁ -Antitrypsin Portland

α₁ -antitrypsin and variants Pittsburgh and Portland were assayed forthe ability to inhibit furin endoprotease in vitro essentially asdescribed in Molloy et al. (ibid.). Briefly, 25 μL of the resuspendedfurin preparation described in Example 1 was incubated with each of theα₁ -antitrypsins (at a final concentration of 10 μg/mL) for 20 min at37° C. in a buffer comprising 100 mM HEPES (pH 7.5), 1 mM CaCl₂, 1 mM2-mercaptoethanol and 0.5% Triton-X 100. Substrate((N-butoxycarbonyl)-Arg-Val-Arg-(4-methylcoumaryl-7-amide); PeninsulaLaboratories, Belmont, Calif.) was then added to a final concentrationof 50 μM and incubated for 30 min at 37° C. The amount of liberatedaminomethylcoumarin was then determined by fluorimetry (excitationwavelength=380 nm; emission wavelength=460 nm) using aspectrofluorimeter (Perkin Elmer, Waterbury, Conn. Model LS3). Theresults of this experiment are shown in FIG. 2. For comparison, each ofthe α₁ -antitrypsins were also incubated with thrombin and assayed forthrombin peptidase activity colorometrically usingbenzoyl-Phe-Val-Arg-(para-nitroanilide) as substrate. These results arealso shown in FIG. 2. Native α₁ -antitrypsin was unable to inhibiteither furin or thrombin endoprotease activity (striped

                  TABLE I                                                         ______________________________________                                        Enzyme    Conc..sup.a                                                                           α.sub.1 -NAT.sup.b                                                                 α.sub.1 -PIT.sup.b                                                            α.sub.1 -PDX.sup.b                   ______________________________________                                        Elastase  100     13         98    101                                        Thrombin  100     94         8     95                                         Furin     100     101        97    0.04                                       ______________________________________                                         .sup.a = relative concentration of each serpin inhibitor                      .sup.b = percent endoprotease activity in presence of each serpin        

bars). α₁ -antitrypsin Pittsburgh specifically inhibited thrombin butnot furin (dotted bars). The novel variant α₁ -antitrypsin Portland(stipled bars) had no effect on thrombin endoprotease activity, butspecifically and essentially quantitatively inhibited furin endoproteaseactivity. Table I provides the results of a comparison between thelevels of inhibition of elastase (assayed colorometrically with(N-butoxycarbonyl)-Ala-Ala-Pro-Ala-(para-nitroanilide) as substrate).Elastase activity was specifically inhibited by native α₁ -antitrypsin(α₁ -NAT), thrombin was specifically inhibited by α₁ -antitrypsinPittsburgh (α₁ -PIT), and furin was specifically inhibited by α₁-antitrypsin Portland (α₁ -PDX). α₁ -PDX was found to be greater than300-fold more effective than α₁ -PIT in inhibiting furin, having aK_(1/2) of 0.03 μg/mL, (equivalent to 0.4 nM). α₁ -PDX is also greatlyattenuated in thrombin inhibitory activity (>300-fold) compared with α₁-PIT. These results demonstrated that the novel α₁ -antitrypsinstructural variant (α₁ -PDX) disclosed herein has novel functionalproperties that make it useful as a specific furin endoproteaseinhibitor.

EXAMPLE 4 In Vivo Characterization of Furin Endoprotease Inhibition byα₁ -Antitrypsin Portland

α₁ -antitrypsin Portland was assayed for the ability to inhibit furinendoprotease in vivo. BSC-40 cells were infected with a vaccinia virusvector encoding the α₁ -antitrypsin Portland as described in Example 2and a vaccinia virus vector encoding pro-β-nerve growth factor (β-NGF),a neuropeptide growth factor known to be processed by furin at theconsensus furin site -Arg-Ser-Lys-Arg↓- (SEQ ID NO.: 1) (Bresnahan etal., ibid.) and secreted into the cell growth media. Co-infected cellswere incubated in the presence of (³⁵ S)-methionine for 4 h afterinfection and the cell media harvested. Media samples were thenimmunoprecipitated with NGF-specific antibodies and assayed by sodiumdodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) andvisualized by fluorography using standard techniques (as described inSambrook et al., ibid.)

The results of these experiments are shown in FIG. 3. Cells infectedwith wild-type vaccinia virus secreted no detectable β-NGF into theculture media (Lane A), whereas cells infected with the vaccinia virusvector encoding β-NGF (Lane B) secreted both processed (13 kilodaltons,kD) and unprocessed (≈35 kD) forms of β-NGF; co-infection of such cellswith wild-type vaccinia virus had no effect on this pattern of β-NGFproduction (Lane C). Similarly, BSC-40 cells co-infected with the β-NGFconstruct and with vaccinia virus constructs encoding native α₁-antitrypsin (Lane D) and α₁ -antitrypsin Pittsburgh (Lane E) alsoproduced both the processed and unprocessed forms of β-NGF. In contrast,cells co-infected with the β-NGF construct and with vaccinia virusconstructs encoding α₁ -antitrypsin Portland (Lane F) produced only theunprocessed form of β-NGF, demonstrating that α₁ -antitrypsin Portlandis capable of inhibiting furin-mediated endoprotease processing ofbioactive pro-peptides in vivo.

EXAMPLE 5 Inhibition of Furin-Mediated Processing of HumanImmunodeficiency Virus gp160 by α₁ -Antitrypsin Portland

The experiments described in Example 4 were repeated using a vacciniavirus construct encoding the Human Immunodeficiency virus (HIV-1)glycoprotein gp160. This precursor protein is known to beproteolytically processed into two membrane-associated proteins: gp120(which binds the HIV receptor CD4 on the cell surface of target hostcells) and gp41 (which provides a fusogenic activity that mediates viralentry into the cell) in vivo. Proteolytic processing at the furinconsensus site -Arg-Glu-Lys-Arg↓- (SEQ ID NO.: 3) is a necessary step inmaturation and release of HIV viral particles. Cell membranes from cellsinfected with vaccinia virus constructs were isolated as described inExample 1. Proteins from such membrane preparations were resolved bySDS-PAGE and specifically identified by Western blot analysis (seeSambrook et al., ibid., Chapter 18) using antibodies against HIVproteins (see Anderson et al., supra).

The results of this experiment are shown in FIG. 4. Cells infected withwild-type vaccinia virus produced no detectable HIV-related peptides(Lane A). In cells infected with the vaccinia virus vector encodinggp160, both the unprocessed protein and processing products, gp120 andgp41 were produced (Lane B). BSC-40 cells co-infected with the gp160construct and with vaccinia virus constructs encoding native α₁-antitrypsin (Lane C) and α₁ -antitrypsin Pittsburgh (Lane D) alsoproduced both unprocessed gp160 and processed gp120 and gp41. Cellsco-infected with the gp160 construct and with vaccinia virus constructsencoding α₁ -antitrypsin Portland (Lane E), on the other hand, producedonly unprocessed gp160. These results demonstrated that α₁ -antitrypsinPortland is capable of inhibiting furin-mediated endoprotease processingof bioactive viral proteins in vivo, and immediately suggested a methodfor treating viral infection by inhibiting viral protein processing.

In another series of experiments, α₁ -antitrypsin Portland-mediatedinhibition of proteolytic processing of gp160 was assayed to determinethe functional consequences of such inhibition. As described above,processing of gp160 results in the production of gp41, a protein thatprovides a fusogenic activity important for viral entry into targetcells. Expression of gp120 and gp41 on the surface of IP/IRerythroleukemia cells (see Spiro et al., 1988, J. Virol. 63: 4434-4437)promotes cell fusion and syncytium formation with cells expressing thegp120 target, CD4, at the cell surface. In these experiments, IP/IRcells were co-infected with vaccinia virus expression constructsencoding gp160 and each of the α₁ -antitrypsins to determine the effectof inhibition of gp160 processing on the fusogenic capacity of thecells. This experimental protocol is shown in FIG. 5. Briefly, IP/IRcells were co-infected with the vaccinia virus gp160 construct, eitheralone or co-infected with each of the α₁ -antitrypsin constructsdescribed in Example 2. The cells were incubated for 8-16 hours,collected and then overlaid onto a monolayer of CD4⁺ human HeLa cells amodification of parental HeLa cells (ATCC No. CCL 2) that express theCD4 cell surface protein, as described in Kabat et al., 1994, Virol.202: 1058-1060. These cells were incubated for an additional 8-12 hours,and syncytium formation detected by staining with crystal violet andobserved by phase-contract microscopy.

The results of these experiments are shown in FIGS. 6A through 6F. FIG.6A shows the results of HeLa/CD4⁺ cell overlay with IP/IR cells infectedwith vaccinia virus recombinants encoding HIV gp160, and FIG. 6B showsresults of HeLa/CD4⁺ cell overlay experiments using IP/IR cells infectedwith HIV gp160 vaccinia recombinants co-infected with wild type vacciniavirus. Syncytia formation (and hence proper proteolytic processing ofgp160 to gp120 and gp41) in the cells of each of these infectedco-cultures was evidenced by the large number of multinucleated cells ineach of the cultures. FIG. 6C shows the results of HeLa/CD4⁺ celloverlay experiments using IP/IR cells infected with wild-type vacciniavirus; essentially no multinucleated cells were seen in such cultures.FIGS. 6D and 6E show the results of HeLa CD4⁺ cell overlay experimentsusing IP/IR cells co-infected with vaccinia virus recombinants encodinggp160 and native (FIG. 6C) and the Pittsburgh variant (FIG. 6D) of α₁-antitrypsin. Co-expression of native or variant Pittsburgh α₁-antitrypsin had no effect on syncytia formation caused by gp41. FIG. 6Fshows the effect on HIV gp41-mediated syncytia formation of HeLa CD4⁺cell overlay experiments using IP/IR cells co-infected with vacciniavirus recombinants encoding gp160 and α₁ -antitrypsin Portland inHeLa/CD4⁺ cells. Syncytia formation is completely abolished in thesecultures, and the cells of such cultures looked identical to cells inoverlay experiments using IP/IR cells infected with wild-type vacciniavirus as seen in FIG. 6C. These results demonstrate that inhibition ofgp160 processing by α₁ -antitrypsin Portland eliminates the fusogenicactivity of viral gp41 and suggests that such inhibition may provide amethod for treating HIV infection in vivo and in vitro.

EXAMPLE 6 Inhibition of Furin-Mediated Processing of β-Nerve GrowthFactor by Direct Application of α₁ -Antitrypsin Portland

The ability of α₁ -antitrypsin Portland to inhibit proteolyticprocessing by addition of the inhibitor to the cellular growth media wasdemonstrated in the following assay. BSC-40 cells were grown toconfluence in 35 mm tissue culture plates (Falcon Microbiological SupplyCo., Lincoln Park, N.J.) and then infected with recombinant vacciniavirus encoding a functional pro-β-NGF protein (VV:mNGF) as described inExample 4. Four hours prior to infection, the cellular growth media wasremoved and replaced with serum-free defined media (MCDB 202 media, madeas described in McKeeton et al., 1977, Dev. Biol. Std. 37: 97-108)supplemented with either 0, 0.5 or 50 μg/mL α₁ -antitrypsin Portland (α₁-PDX), prepared as described in Example 2. After this 4 h incubation,the cells were infected with VV:mNGF at a multiplicity of infection(m.o.i.) of 5 pfu/cell. In one culture, the pre-incubated cells weretransfected with VV:mNGF at a m.o.i. of 2 and co-infected with VV:α₁-PDX (see Example 2) at a m.o.i. of 5. After virus inoculation, α₁ -PDXwas added to the culture media of each infected plate at the sameconcentration as in the pre-incubation period.

Eighteen hours post-infection the media was removed from each plate andthe cells metabolically labeled by incubation for 5 h in mediacontaining (³⁵ S)-methionine and (³⁵ S)-cysteine (500 μCi) and α₁ -PDXat the appropriate concentration for each plate. After labeling, theculture media from each plate was harvested and NGF peptidesimmunoprecipitated, resolved by SDS-PAGE and visualized by fluorographyas described in Example 4.

The results of this experiment are shown in FIGS 6A and 7B. FIG. 7A isan autoradiograph of SDS-PAGE analysis of immunoprecipitated NGFpeptides from each of the infected cultures, in which lane 1 containsthe results from the culture incubated with α₁ -PDX at 0 μg/mL; lane 2contains the results from the culture incubated with α₁ -PDX at 0.5μg/mL; lane 3 contains the results from the culture incubated with α₁-PDX at 50 μg/mL; and lane 4 contains the results from the cultureco-infected with VV:α₁ -PDX. As this autoradiographic evidence shows,incubation of cell cultures with α₁ -PDX protein resulted in markedattenuation of furin-directed proteolytic processing of pro-β-NGF in adose-dependent manner. The autoradiographic data was quantitated bydensitometry as shown in FIG. 7B; the percent relative amount ofinhibition of proteolytic processing is represented as a percentage ofthe density of the processed (β-NGF) band relative to the density of thesum of the processed plus the unprocessed (pro-β-NGF) band. FIGS. 6A and7B shows that α₁ -PDX at 50 μg/mL reduces the amount of proteolyticprocessing about five-fold (47% (0 μg/mL) versus 10% (50 μg/mL)).Essentially no processing is seen in the co-infected cell line,consistent with the results described in Example 4 above.

This dose-dependent attenuation of furin-directed proteolytic processingfollowing administration of α₁ -PDX protein directly to cells in culturedemonstrates the feasibility of a protein-based therapeutic approachdirected at inhibiting furin-directed proteolytic maturation of avariety of biologically-important protein molecules, which isadditionally advantageous due to the lack of thrombin inhibitionexhibited by α₁ -PDX protein as disclosed herein.

EXAMPLE 7 Construction of an HIV-LTR-based Recombinant Expression VectorEncoding α₁ -Antitrypsin Portland

In order to stably transfect cells with a recombinant expressionconstruct capable of expressing α₁ -PDX protein in vivo, the followingexpression vector was made, wherein expression of α₁ -PDX was mediatedby the long terminal repeat (LTR) sequences of HIV-2. The completedplasmid is shown in FIG. 8.

A 470 bp fragment was excised from the recombinant plasmid pGEM/RRE bySmaI/HindIII digestion and then blunt-end cloned into the BamHI site ofthe plasmid pRep4 (Invitrogen, San Diego, Calif.). Restrictionenzyme-generated overhangs were blunt-ended using Klenow polymerase(Pharmacia, Upsala, Sweden) using conventional techniques (see Sambrooket al.). The 470 bp fragment contains the rev responsive element ofHIV-HXB2, comprising nucleotides 7621-8140 (Malim et al., 1989, Nature338: 254-257). The Rous Sarcoma Virus 3' LTR was excised from theresulting plasmid by a SalI (partial blunt)/HindIII digest, and replacedby ligation with an EcoRI(blunt)/HindIII digestion fragment frompBennCAT (NIH AIDS Research Program, Bethesda, Md.) containing the HIVLTR (nucleotides -450 to +80). The resulting expression plasmid wastermed pRep4/RRE.

Plasmids capable of expressing α₁ -PIT or α₁ -PDX cDNAs were constructedby inserting each of the cDNA molecules into pRep4/RRE between the HIVLTR and the RRE sequence in the proper orientation. This was done byexcising α₁ -PIT cDNA sequences from pA1AT PIT (Long et al., supra)using EcoRI (blunt) and XhoI digestion, or by excising α₁ -PDX cDNAsequences from pAIAT PDX using SmaI and XhoI digestion, and cloning eachcDNA into HindIII (blunt)/XhaI-digested pRep4/RRE. In a final step, theEpstein Barr virus origin of replication (ORIP) and nuclear antigen(EBNA-1) sequences were excised from the final versions of each of theα₁ -antitrypsin variant expression constructs as follows. pRep4/RRE wasdigested with SpeI and ClaI, blunt-ended as described above, andreligated to itself (see FIG. 8). A XbaI/SacII fragment from thepRep4/RRE plasmid so modified was then swapped for the correspondingfragment in each of the α₁ -antitrypsin variant plasmids describedabove, This resulted in plasmids having the structure illustrated inFIG. 8 and encoding each of the α₁ -antitrypsin variants α₁ -PIT(plasmid pRep4ΔL/Rpα₁ -PIT) and α₁ -PDX (plasmid pRep4ΔL/Rpα₁ -PDX).

EXAMPLE 8 Establishment of Human CD4⁺ Cell Lines Expressing α₁-Antitrypsin Portland

The pRep4ΔL/Rpα₁ -PDX plasmid contains a functional hygromycinresistance gene (see FIG. 8) capable of conferring hygromycin resistanceto eukaryotic cells (see Product Catalog, Invitrogen). Ten micrograms ofthis plasmid DNA were introduced into HeLa/CD4⁺ clone 1022 cells(described in Example 5 above) using a modified calcium phosphateprecipitation technique (Chen & Okayama, 1988, Molec. Cell. Biol. 8:123-130, as further described in Sambrook et al., ibid.). Two days aftertransfection the cells were placed on selective media RPMI60/10% fetalcalf serum supplemented with 100 μg/mL hygromycin (Sigma Chemical Co.)and maintained in this media throughout the experiment. Cell clonesresistant to the drug appeared after about two weeks in selective media,and individual clones were isolated and expanded for subsequentexperiments using conventional techniques (see Tissue Culture, AcademicPress, Kruse & Patterson, editors (1973)). Two such clones (termed PDX-4and PDX-6) were used in the following experiments.

Cell lines PDX-4, PDX-6 and parental HeLa CD4⁺ clone 1022 were seeded in6-well plates (Falcon) at a density of 2×10⁵ cells/plate. Cells cultureswere then infected in duplicate with 2 mL of media containing HIV strainLAV (NIH AIDS Research Program) having a reverse transcriptase activityof about 70,000 cpm/mL. Cell cultures were examined four dayspost-infection for cytopathic effects and syncytia formation. Suchcultures are shown in FIGS. 9A through 9C. The parental HeLa clone 1022cells (FIG. 9B) exhibited numerous syncytia and cytopathic loss of thecell monolayer. Both PDX-4 (FIG. 9C) and PDX-6 (FIG. 9A) cell cultures,on the other hand, showed few syncytia. Productive infection wasverified by the detection of more than 250 pg/mL of the HIV coreantigen, p24, in supernatants from each of the infected cultures. Thelevels of infectious virus, in contrast, were much lower in the pDX-4and PDX-6 cultures than in the parental HeLa clone 1022 culture. Celllysates were prepared from each of the cell cultures on the fourth daypost-infection and examined for the presence of gp160/gp120 Western blotanalysis as described in Example 5. Both gp160 and bp120 were detectedin HIV-infected HeLa clone 1022 cultures, while no gp120 and only afaint band corresponding to gp160 was found in HIV-infected PDX-4 andPDX-6 cultures.

These results demonstrated that expression of α₁ -PDX in HeLa clone 1022cells conferred resistance in these cells to HIV infection. Moreover,the presence of p24 antigen in the cell supernatants of such cultures inthe absence of large amounts of infectious virus indicates thatexpression of α₁ -antitrypsin Portland in these cells inhibits theformation of infectious enveloped virus particles. These results haveimportant implications for preventing HIV infection of vulnerable cellsboth in vivo and in vitro.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 21                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Arg Ser Lys Arg                                                             - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Arg Pro Lys Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Arg Glu Lys Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Arg Val Arg Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #site     (A) NAME/KEY: Modified                                                        (B) LOCATION: 2..3                                                  #/label=Variable siteNFORMATION:                                                             / note=- #"The amino acid Xaa at position 2 can be                            any amino - # acid; the amino acid Xaa at position 3           #any amino acid."n be                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Arg Xaa Xaa Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 394 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Th - #r Asp Thr Ser His His         #                15                                                           - Asp Gln Asp His Pro Thr Phe Asn Lys Ile Th - #r Pro Asn Leu Ala Glu         #            30                                                               - Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala Hi - #s Gln Ser Asn Ser Thr         #        45                                                                   - Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Th - #r Ala Phe Ala Met Leu         #    60                                                                       - Ser Leu Gly Thr Lys Ala Asp Thr His Asp Gl - #u Ile Leu Glu Gly Leu         #80                                                                           - Asn Phe Asn Leu Thr Gln Ile Pro Glu Ala Gl - #n Ile His Glu Gly Phe         #                95                                                           - Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro As - #p Ser Gln Leu Gln Leu         #           110                                                               - Thr Thr Gly Asn Gly Leu Phe Leu Ser Gln Gl - #y Leu Lys Leu Val Asp         #       125                                                                   - Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr Hi - #s Ser Glu Ala Phe Thr         #   140                                                                       - Val Asn Phe Gly Asp Thr Glu Gln Ala Lys Ly - #s Gln Ile Asn Asp Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Glu Lys Gly Thr Gln Gly Lys Ile Val As - #p Leu Val Lys Glu Leu         #               175                                                           - Asp Arg Asp Thr Val Phe Ala Leu Val Asn Ty - #r Ile Phe Phe Lys Gly         #           190                                                               - Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Th - #r Glu Glu Glu Asp Phe         #       205                                                                   - His Val Asp Gln Val Thr Thr Val Lys Val Pr - #o Met Met Lys Arg Leu         #   220                                                                       - Gly Met Phe Asn Ile Gln His Cys Lys Lys Le - #u Ser Ser Trp Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Il - #e Phe Phe Leu Pro Asp         #               255                                                           - Glu Gly Lys Leu Gln His Leu Glu Asn Glu Le - #u Thr His Asp Ile Ile         #           270                                                               - Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Se - #r Ala Ser Leu His Leu         #       285                                                                   - Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Le - #u Lys Ser Val Leu Gly         #   300                                                                       - Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gl - #y Ala Asp Leu Ser Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Ly - #s Ala Val His Lys Ala         #               335                                                           - Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Al - #a Ala Gly Ala Met Phe         #           350                                                               - Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Gl - #u Val Lys Phe Asn Lys         #       365                                                                   - Pro Phe Val Phe Leu Met Ile Glu Gln Asn Th - #r Lys Ser Pro Leu Phe         #   380                                                                       - Met Gly Lys Val Val Asn Pro Thr Gly Lys                                     385                 3 - #90                                                   - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Ala Ile Pro Met                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #site     (A) NAME/KEY: Modified                                                        (B) LOCATION: 2..3                                                  #/label=Variable siteNFORMATION:                                                             / note=- #"The amino acid Xaa at position 2 can be                            any amino - # acid; the amino acid Xaa at position 3           #any amino acid."n be                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Arg Xaa Xaa Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 394 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #site     (A) NAME/KEY: Modified                                                        (B) LOCATION: 355..358                                              #/label=VariantTHER INFORMATION:                                                             / note=- #"The amino acid sequence is the amino acid           #of the modified alpha-1-antitrypsin                                          #alpha-1-antitrypsin Portland."                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Th - #r Asp Thr Ser His His         #                15                                                           - Asp Gln Asp His Pro Thr Phe Asn Lys Ile Th - #r Pro Asn Leu Ala Glu         #            30                                                               - Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala Hi - #s Gln Ser Asn Ser Thr         #        45                                                                   - Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Th - #r Ala Phe Ala Met Leu         #    60                                                                       - Ser Leu Gly Thr Lys Ala Asp Thr His Asp Gl - #u Ile Leu Glu Gly Leu         #80                                                                           - Asn Phe Asn Leu Thr Gln Ile Pro Glu Ala Gl - #n Ile His Glu Gly Phe         #                95                                                           - Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro As - #p Ser Gln Leu Gln Leu         #           110                                                               - Thr Thr Gly Asn Gly Leu Phe Leu Ser Gln Gl - #y Leu Lys Leu Val Asp         #       125                                                                   - Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr Hi - #s Ser Glu Ala Phe Thr         #   140                                                                       - Val Asn Phe Gly Asp Thr Glu Gln Ala Lys Ly - #s Gln Ile Asn Asp Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Glu Lys Gly Thr Gln Gly Lys Ile Val As - #p Leu Val Lys Glu Leu         #               175                                                           - Asp Arg Asp Thr Val Phe Ala Leu Val Asn Ty - #r Ile Phe Phe Lys Gly         #           190                                                               - Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Th - #r Glu Glu Glu Asp Phe         #       205                                                                   - His Val Asp Gln Val Thr Thr Val Lys Val Pr - #o Met Met Lys Arg Leu         #   220                                                                       - Gly Met Phe Asn Ile Gln His Cys Lys Lys Le - #u Ser Ser Trp Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Il - #e Phe Phe Leu Pro Asp         #               255                                                           - Glu Gly Lys Leu Gln His Leu Glu Asn Glu Le - #u Thr His Asp Ile Ile         #           270                                                               - Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Se - #r Ala Ser Leu His Leu         #       285                                                                   - Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Le - #u Lys Ser Val Leu Gly         #   300                                                                       - Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gl - #y Ala Asp Leu Ser Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Ly - #s Ala Val His Lys Ala         #               335                                                           - Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Al - #a Ala Gly Ala Met Phe         #           350                                                               - Leu Glu Arg Ile Pro Arg Ser Ile Pro Pro Gl - #u Val Lys Phe Asn Lys         #       365                                                                   - Pro Phe Val Phe Leu Met Ile Glu Gln Asn Th - #r Lys Ser Pro Leu Phe         #   380                                                                       - Met Gly Lys Val Val Asn Pro Thr Gly Lys                                     385                 3 - #90                                                   - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Arg Ile Pro Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 394 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #site     (A) NAME/KEY: Modified                                                        (B) LOCATION: 355..358                                              #/label=VariantTHER INFORMATION:                                                             / note=- #"The amino acid sequence is the amino acid           #of the modified alpha-1-antitrypsin                                          #alpha-1-antitrypsin Pittsburgh."                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Th - #r Asp Thr Ser His His         #                15                                                           - Asp Gln Asp His Pro Thr Phe Asn Lys Ile Th - #r Pro Asn Leu Ala Glu         #            30                                                               - Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala Hi - #s Gln Ser Asn Ser Thr         #        45                                                                   - Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Th - #r Ala Phe Ala Met Leu         #    60                                                                       - Ser Leu Gly Thr Lys Ala Asp Thr His Asp Gl - #u Ile Leu Glu Gly Leu         #80                                                                           - Asn Phe Asn Leu Thr Gln Ile Pro Glu Ala Gl - #n Ile His Glu Gly Phe         #                95                                                           - Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro As - #p Ser Gln Leu Gln Leu         #           110                                                               - Thr Thr Gly Asn Gly Leu Phe Leu Ser Gln Gl - #y Leu Lys Leu Val Asp         #       125                                                                   - Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr Hi - #s Ser Glu Ala Phe Thr         #   140                                                                       - Val Asn Phe Gly Asp Thr Glu Gln Ala Lys Ly - #s Gln Ile Asn Asp Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Glu Lys Gly Thr Gln Gly Lys Ile Val As - #p Leu Val Lys Glu Leu         #               175                                                           - Asp Arg Asp Thr Val Phe Ala Leu Val Asn Ty - #r Ile Phe Phe Lys Gly         #           190                                                               - Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Th - #r Glu Glu Glu Asp Phe         #       205                                                                   - His Val Asp Gln Val Thr Thr Val Lys Val Pr - #o Met Met Lys Arg Leu         #   220                                                                       - Gly Met Phe Asn Ile Gln His Cys Lys Lys Le - #u Ser Ser Trp Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Il - #e Phe Phe Leu Pro Asp         #               255                                                           - Glu Gly Lys Leu Gln His Leu Glu Asn Glu Le - #u Thr His Asp Ile Ile         #           270                                                               - Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Se - #r Ala Ser Leu His Leu         #       285                                                                   - Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Le - #u Lys Ser Val Leu Gly         #   300                                                                       - Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gl - #y Ala Asp Leu Ser Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Ly - #s Ala Val His Lys Ala         #               335                                                           - Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Al - #a Ala Gly Ala Met Phe         #           350                                                               - Leu Glu Ala Ile Pro Arg Ser Ile Pro Pro Gl - #u Val Lys Phe Asn Lys         #       365                                                                   - Pro Phe Val Phe Leu Met Ile Glu Gln Asn Th - #r Lys Ser Pro Leu Phe         #   380                                                                       - Met Gly Lys Val Val Asn Pro Thr Gly Lys                                     385                 3 - #90                                                   - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1356 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 34..1275                                              #/product= alpha-1-antitrypsinN:                                                             variant P - #ittsburgh"                                                       / standar - #d.sub.-- name= alpha-1-antitrypsin Pittsburgh     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - GCACCACCAC TGACCTGGGA CAGTGAATCG ACA ATG CCG TCT TC - #T GTC TCG TGG          54                                                                          # Met Pro Ser Ser Val Ser Trp                                                 #5  1                                                                         - GGC ATC CTC CTG CTG GCA GGC CTG TGC TGC CT - #G GTC CCT GTC TCC CTG          102                                                                          Gly Ile Leu Leu Leu Ala Gly Leu Cys Cys Le - #u Val Pro Val Ser Leu           #         20                                                                  - GCT GAG GAT CCC CAG GGA GAT GCT GCC CAG AA - #G ACA GAT ACA TCC CAC          150                                                                          Ala Glu Asp Pro Gln Gly Asp Ala Ala Gln Ly - #s Thr Asp Thr Ser His           #     35                                                                      - CAT GAT CAG GAT CAC CCA ACC TTC AAC AAG AT - #C ACC CCC AAC CTG GCT          198                                                                          His Asp Gln Asp His Pro Thr Phe Asn Lys Il - #e Thr Pro Asn Leu Ala           # 55                                                                          - GAG TTC GCC TTC AGC CTA TAC CGC CAG CTG GC - #A CAC CAG TCC AAC AGC          246                                                                          Glu Phe Ala Phe Ser Leu Tyr Arg Gln Leu Al - #a His Gln Ser Asn Ser           #                 70                                                          - ACC AAT ATC TTC TTC TCC CCA GTG AGC ATC GC - #T ACA GCC TTT GCA ATG          294                                                                          Thr Asn Ile Phe Phe Ser Pro Val Ser Ile Al - #a Thr Ala Phe Ala Met           #             85                                                              - CTC TCC CTG GGG ACC AAG GCT GAC ACT CAC GA - #T GAA ATC CTG GAG GGC          342                                                                          Leu Ser Leu Gly Thr Lys Ala Asp Thr His As - #p Glu Ile Leu Glu Gly           #        100                                                                  - CTG AAT TTC AAC CTC ACG GAG ATT CCG GAG CC - #T CAG ATC CAT GAA GGC          390                                                                          Leu Asn Phe Asn Leu Thr Glu Ile Pro Glu Pr - #o Gln Ile His Glu Gly           #   115                                                                       - TTC CAG GAA CTC CTC CGT ACC CTC AAC CAG CT - #C CAG CTG ACC ACC GGC          438                                                                          Phe Gln Glu Leu Leu Arg Thr Leu Asn Gln Le - #u Gln Leu Thr Thr Gly           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - AAT GGC CTG TTC CTC AGC GAG GGC CTG AAG CT - #A GTG GAT AAG TTT TTG          486                                                                          Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Le - #u Val Asp Lys Phe Leu           #               150                                                           - GAG GAT GTT AAA AAG TTG TAC CAC TCA GAA GC - #C TTC ACT GTC AAC TTC          534                                                                          Glu Asp Val Lys Lys Leu Tyr His Ser Glu Al - #a Phe Thr Val Asn Phe           #           165                                                               - GGG GAC ACC GAA GAG GCC AAG AAA CAG ATC AA - #C GAT TAC GTG GAG AAG          582                                                                          Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile As - #n Asp Tyr Val Glu Lys           #       180                                                                   - GGT ACT CAA GGG AAA ATT GTG GAT TTG GTC AA - #G GAG CTT GAC AGA GAC          630                                                                          Gly Thr Gln Gly Lys Ile Val Asp Leu Val Ly - #s Glu Leu Asp Arg Asp           #   195                                                                       - ACA GTT TTT GCT CTG GTG AAT TAC ATC TTC TT - #T AAA GGC AAA TGG GAG          678                                                                          Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Ph - #e Lys Gly Lys Trp Glu           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - AGA CCC TTT GAA GTC AAG GAC ACC GAG GAA GA - #G GAC TTC CAC GTG GAC          726                                                                          Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Gl - #u Asp Phe His Val Asp           #               230                                                           - CAG GTG ACC ACC GTG AAG GTG CCT ATG ATG AA - #G CGT TTA GGC ATG TTT          774                                                                          Gln Val Thr Thr Val Lys Val Pro Met Met Ly - #s Arg Leu Gly Met Phe           #           245                                                               - AAC ATC CAG CAC TGT AAG AAG CTG TCC AGC TG - #G GTG CTG CTG ATG AAA          822                                                                          Asn Ile Gln His Cys Lys Lys Leu Ser Ser Tr - #p Val Leu Leu Met Lys           #       260                                                                   - TAC CTG GGC AAT GCC ACC GCC ATG TTC TTC CT - #G CCT GAT GAG GGG AAA          870                                                                          Tyr Leu Gly Asn Ala Thr Ala Met Phe Phe Le - #u Pro Asp Glu Gly Lys           #   275                                                                       - CTA CAG CAC CTG GAA AAT GAA CTC ACC CAC GA - #T ATC ATC ACC AAG TTC          918                                                                          Leu Gln His Leu Glu Asn Glu Leu Thr His As - #p Ile Ile Thr Lys Phe           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - CTG GAA AAT GAA GAC AGA AGG TCT GCC AGC TT - #A CAT TTA CCC AAA CTG          966                                                                          Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Le - #u His Leu Pro Lys Leu           #               310                                                           - TCC ATT ACT GGA ACC TAT GAT CTG AAG AGC GT - #C CTG GGT CAA CTG GGC         1014                                                                          Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Va - #l Leu Gly Gln Leu Gly           #           325                                                               - ATC ACT AAG GTC TTC AGC AAT GGG GCT GAC CT - #C TCC GGG GTC ACA GAG         1062                                                                          Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Le - #u Ser Gly Val Thr Glu           #       340                                                                   - GAG GCA CCC CTG AAG CTC TCC AAG GCC GTG CA - #T AAG GCT GTG CTG ACC         1110                                                                          Glu Ala Pro Leu Lys Leu Ser Lys Ala Val Hi - #s Lys Ala Val Leu Thr           #   355                                                                       - ATC GAC GAG AAA GGG ACT GAA GCT GCT GGG GC - #C ATG TTT TTA GAG GCC         1158                                                                          Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Al - #a Met Phe Leu Glu Ala           360                 3 - #65                 3 - #70                 3 -       #75                                                                           - ATA CCC AGG TCT ATC CCC CCC GAG GTC AAG TT - #C AAC AAA CCC TTT GTC         1206                                                                          Ile Pro Arg Ser Ile Pro Pro Glu Val Lys Ph - #e Asn Lys Pro Phe Val           #               390                                                           - TTC TTA ATG ATT GAA CAA AAT ACC AAG TCT CC - #C CTC TTC ATG GGA AAA         1254                                                                          Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pr - #o Leu Phe Met Gly Lys           #           405                                                               - GTG GTG AAT CCC ACC CAA AAA TAACTGCTCG CTCCTCAAC - #C CCTCCCCTCC            1305                                                                          Val Val Asn Pro Thr Gln Lys                                                           410                                                                   #           1356TCCCTGG ATGACATTAA AGAAGGGTTG AGCTGGAAAA A                    - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 414 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Met Pro Ser Ser Val Ser Trp Gly Ile Leu Le - #u Leu Ala Gly Leu Cys         #                 15                                                          - Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pr - #o Gln Gly Asp Ala Ala         #             30                                                              - Gln Lys Thr Asp Thr Ser His His Asp Gln As - #p His Pro Thr Phe Asn         #         45                                                                  - Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Ph - #e Ser Leu Tyr Arg Gln         #     60                                                                      - Leu Ala His Gln Ser Asn Ser Thr Asn Ile Ph - #e Phe Ser Pro Val Ser         # 80                                                                          - Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gl - #y Thr Lys Ala Asp Thr         #                 95                                                          - His Asp Glu Ile Leu Glu Gly Leu Asn Phe As - #n Leu Thr Glu Ile Pro         #           110                                                               - Glu Pro Gln Ile His Glu Gly Phe Gln Glu Le - #u Leu Arg Thr Leu Asn         #       125                                                                   - Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Ph - #e Leu Ser Glu Gly Leu         #   140                                                                       - Lys Leu Val Asp Lys Phe Leu Glu Asp Val Ly - #s Lys Leu Tyr His Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Gl - #u Glu Ala Lys Lys Gln         #               175                                                           - Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gl - #y Lys Ile Val Asp Leu         #           190                                                               - Val Lys Glu Leu Asp Arg Asp Thr Val Phe Al - #a Leu Val Asn Tyr Ile         #       205                                                                   - Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Gl - #u Val Lys Asp Thr Glu         #   220                                                                       - Glu Glu Asp Phe His Val Asp Gln Val Thr Th - #r Val Lys Val Pro Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Lys Arg Leu Gly Met Phe Asn Ile Gln Hi - #s Cys Lys Lys Leu Ser         #               255                                                           - Ser Trp Val Leu Leu Met Lys Tyr Leu Gly As - #n Ala Thr Ala Met Phe         #           270                                                               - Phe Leu Pro Asp Glu Gly Lys Leu Gln His Le - #u Glu Asn Glu Leu Thr         #       285                                                                   - His Asp Ile Ile Thr Lys Phe Leu Glu Asn Gl - #u Asp Arg Arg Ser Ala         #   300                                                                       - Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gl - #y Thr Tyr Asp Leu Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Va - #l Phe Ser Asn Gly Ala         #               335                                                           - Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Le - #u Lys Leu Ser Lys Ala         #           350                                                               - Val His Lys Ala Val Leu Thr Ile Asp Glu Ly - #s Gly Thr Glu Ala Ala         #       365                                                                   - Gly Ala Met Phe Leu Glu Ala Ile Pro Arg Se - #r Ile Pro Pro Glu Val         #   380                                                                       - Lys Phe Asn Lys Pro Phe Val Phe Leu Met Il - #e Glu Gln Asn Thr Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Pro Leu Phe Met Gly Lys Val Val Asn Pr - #o Thr Gln Lys                 #               410                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:single                                                       (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                # 20               CCAG                                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Arg Ile Pro Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1356 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 34..1275                                              #/product= alpha-1-antitrypsinN:                                              #sequence"     wild-type                                                                     / standar - #d.sub.-- name= alpha-1-antitrypsin                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - CACCACCAC TGACCTGGGA CAGTGAATCG ACA ATG CCG TCT TCT - # GTC TCG TGG           54                                                                          #Met Pro Ser Ser Val Ser Trp                                                  # 1               5                                                           - GGC ATC CTC CTG CTG GCA GGC CTG TGC TGC CT - #G GTC CCT GTC TCC CTG          102                                                                          Gly Ile Leu Leu Leu Ala Gly Leu Cys Cys Le - #u Val Pro Val Ser Leu           #         20                                                                  - GCT GAG GAT CCC CAG GGA GAT GCT GCC CAG AA - #G ACA GAT ACA TCC CAC          150                                                                          Ala Glu Asp Pro Gln Gly Asp Ala Ala Gln Ly - #s Thr Asp Thr Ser His           #     35                                                                      - CAT GAT CAG GAT CAC CCA ACC TTC AAC AAG AT - #C ACC CCC AAC CTG GCT          198                                                                          His Asp Gln Asp His Pro Thr Phe Asn Lys Il - #e Thr Pro Asn Leu Ala           # 55                                                                          - GAG TTC GCC TTC AGC CTA TAC CGC CAG CTG GC - #A CAC CAG TCC AAC AGC          246                                                                          Glu Phe Ala Phe Ser Leu Tyr Arg Gln Leu Al - #a His Gln Ser Asn Ser           #                 70                                                          - ACC AAT ATC TTC TTC TCC CCA GTG AGC ATC GC - #T ACA GCC TTT GCA ATG          294                                                                          Thr Asn Ile Phe Phe Ser Pro Val Ser Ile Al - #a Thr Ala Phe Ala Met           #             85                                                              - CTC TCC CTG GGG ACC AAG GCT GAC ACT CAC GA - #T GAA ATC CTG GAG GGC          342                                                                          Leu Ser Leu Gly Thr Lys Ala Asp Thr His As - #p Glu Ile Leu Glu Gly           #        100                                                                  - CTG AAT TTC AAC CTC ACG GAG ATT CCG GAG CC - #T CAG ATC CAT GAA GGC          390                                                                          Leu Asn Phe Asn Leu Thr Glu Ile Pro Glu Pr - #o Gln Ile His Glu Gly           #   115                                                                       - TTC CAG GAA CTC CTC CGT ACC CTC AAC CAG CT - #C CAG CTG ACC ACC GGC          438                                                                          Phe Gln Glu Leu Leu Arg Thr Leu Asn Gln Le - #u Gln Leu Thr Thr Gly           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - AAT GGC CTG TTC CTC AGC GAG GGC CTG AAG CT - #A GTG GAT AAG TTT TTG          486                                                                          Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Le - #u Val Asp Lys Phe Leu           #               150                                                           - GAG GAT GTT AAA AAG TTG TAC CAC TCA GAA GC - #C TTC ACT GTC AAC TTC          534                                                                          Glu Asp Val Lys Lys Leu Tyr His Ser Glu Al - #a Phe Thr Val Asn Phe           #           165                                                               - GGG GAC ACC GAA GAG GCC AAG AAA CAG ATC AA - #C GAT TAC GTG GAG AAG          582                                                                          Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile As - #n Asp Tyr Val Glu Lys           #       180                                                                   - GGT ACT CAA GGG AAA ATT GTG GAT TTG GTC AA - #G GAG CTT GAC AGA GAC          630                                                                          Gly Thr Gln Gly Lys Ile Val Asp Leu Val Ly - #s Glu Leu Asp Arg Asp           #   195                                                                       - ACA GTT TTT GCT CTG GTG AAT TAC ATC TTC TT - #T AAA GGC AAA TGG GAG          678                                                                          Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Ph - #e Lys Gly Lys Trp Glu           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - AGA CCC TTT GAA GTC AAG GAC ACC GAG GAA GA - #G GAC TTC CAC GTG GAC          726                                                                          Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Gl - #u Asp Phe His Val Asp           #               230                                                           - CAG GTG ACC ACC GTG AAG GTG CCT ATG ATG AA - #G CGT TTA GGC ATG TTT          774                                                                          Gln Val Thr Thr Val Lys Val Pro Met Met Ly - #s Arg Leu Gly Met Phe           #           245                                                               - AAC ATC CAG CAC TGT AAG AAG CTG TCC AGC TG - #G GTG CTG CTG ATG AAA          822                                                                          Asn Ile Gln His Cys Lys Lys Leu Ser Ser Tr - #p Val Leu Leu Met Lys           #       260                                                                   - TAC CTG GGC AAT GCC ACC GCC ATG TTC TTC CT - #G CCT GAT GAG GGG AAA          870                                                                          Tyr Leu Gly Asn Ala Thr Ala Met Phe Phe Le - #u Pro Asp Glu Gly Lys           #   275                                                                       - CTA CAG CAC CTG GAA AAT GAA CTC ACC CAC GA - #T ATC ATC ACC AAG TTC          918                                                                          Leu Gln His Leu Glu Asn Glu Leu Thr His As - #p Ile Ile Thr Lys Phe           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - CTG GAA AAT GAA GAC AGA AGG TCT GCC AGC TT - #A CAT TTA CCC AAA CTG          966                                                                          Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Le - #u His Leu Pro Lys Leu           #               310                                                           - TCC ATT ACT GGA ACC TAT GAT CTG AAG AGC GT - #C CTG GGT CAA CTG GGC         1014                                                                          Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Va - #l Leu Gly Gln Leu Gly           #           325                                                               - ATC ACT AAG GTC TTC AGC AAT GGG GCT GAC CT - #C TCC GGG GTC ACA GAG         1062                                                                          Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Le - #u Ser Gly Val Thr Glu           #       340                                                                   - GAG GCA CCC CTG AAG CTC TCC AAG GCC GTG CA - #T AAG GCT GTG CTG ACC         1110                                                                          Glu Ala Pro Leu Lys Leu Ser Lys Ala Val Hi - #s Lys Ala Val Leu Thr           #   355                                                                       - ATC GAC GAG AAA GGG ACT GAA GCT GCT GGG GC - #C ATG TTT TTA GAG GCC         1158                                                                          Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Al - #a Met Phe Leu Glu Ala           360                 3 - #65                 3 - #70                 3 -       #75                                                                           - ATA CCC ATG TCT ATC CCC CCC GAG GTC AAG TT - #C AAC AAA CCC TTT GTC         1206                                                                          Ile Pro Met Ser Ile Pro Pro Glu Val Lys Ph - #e Asn Lys Pro Phe Val           #               390                                                           - TTC TTA ATG ATT GAA CAA AAT ACC AAG TCT CC - #C CTC TTC ATG GGA AAA         1254                                                                          Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pr - #o Leu Phe Met Gly Lys           #           405                                                               - GTG GTG AAT CCC ACC CAA AAA TAACTGCTCG CTCCTCAAC - #C CCTCCCCTCC            1305                                                                          Val Val Asn Pro Thr Gln Lys                                                           410                                                                   #           1356TCCCTGG ATGACATTAA AGAAGGGTTG AGCTGGAAAA A                    - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 414 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Met Pro Ser Ser Val Ser Trp Gly Ile Leu Le - #u Leu Ala Gly Leu Cys         #                 15                                                          - Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pr - #o Gln Gly Asp Ala Ala         #             30                                                              - Gln Lys Thr Asp Thr Ser His His Asp Gln As - #p His Pro Thr Phe Asn         #         45                                                                  - Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Ph - #e Ser Leu Tyr Arg Gln         #     60                                                                      - Leu Ala His Gln Ser Asn Ser Thr Asn Ile Ph - #e Phe Ser Pro Val Ser         # 80                                                                          - Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gl - #y Thr Lys Ala Asp Thr         #                 95                                                          - His Asp Glu Ile Leu Glu Gly Leu Asn Phe As - #n Leu Thr Glu Ile Pro         #           110                                                               - Glu Pro Gln Ile His Glu Gly Phe Gln Glu Le - #u Leu Arg Thr Leu Asn         #       125                                                                   - Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Ph - #e Leu Ser Glu Gly Leu         #   140                                                                       - Lys Leu Val Asp Lys Phe Leu Glu Asp Val Ly - #s Lys Leu Tyr His Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Gl - #u Glu Ala Lys Lys Gln         #               175                                                           - Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gl - #y Lys Ile Val Asp Leu         #           190                                                               - Val Lys Glu Leu Asp Arg Asp Thr Val Phe Al - #a Leu Val Asn Tyr Ile         #       205                                                                   - Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Gl - #u Val Lys Asp Thr Glu         #   220                                                                       - Glu Glu Asp Phe His Val Asp Gln Val Thr Th - #r Val Lys Val Pro Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Lys Arg Leu Gly Met Phe Asn Ile Gln Hi - #s Cys Lys Lys Leu Ser         #               255                                                           - Ser Trp Val Leu Leu Met Lys Tyr Leu Gly As - #n Ala Thr Ala Met Phe         #           270                                                               - Phe Leu Pro Asp Glu Gly Lys Leu Gln His Le - #u Glu Asn Glu Leu Thr         #       285                                                                   - His Asp Ile Ile Thr Lys Phe Leu Glu Asn Gl - #u Asp Arg Arg Ser Ala         #   300                                                                       - Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gl - #y Thr Tyr Asp Leu Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Va - #l Phe Ser Asn Gly Ala         #               335                                                           - Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Le - #u Lys Leu Ser Lys Ala         #           350                                                               - Val His Lys Ala Val Leu Thr Ile Asp Glu Ly - #s Gly Thr Glu Ala Ala         #       365                                                                   - Gly Ala Met Phe Leu Glu Ala Ile Pro Met Se - #r Ile Pro Pro Glu Val         #   380                                                                       - Lys Phe Asn Lys Pro Phe Val Phe Leu Met Il - #e Glu Gln Asn Thr Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Pro Leu Phe Met Gly Lys Val Val Asn Pr - #o Thr Gln Lys                 #               410                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1356 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 34..1275                                              #/product= alpha-1-antitrypsinN:                                                             variant P - #ortland"                                                         / standar - #d.sub.-- name= alpha-1-antitrypsin Portland       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - GCACCACCAC TGACCTGGGA CAGTGAATCG ACA ATG CCG TCT TC - #T GTC TCG TGG          54                                                                          # Met Pro Ser Ser Val Ser Trp                                                 #5  1                                                                         - GGC ATC CTC CTG CTG GCA GGC CTG TGC TGC CT - #G GTC CCT GTC TCC CTG          102                                                                          Gly Ile Leu Leu Leu Ala Gly Leu Cys Cys Le - #u Val Pro Val Ser Leu           #         20                                                                  - GCT GAG GAT CCC CAG GGA GAT GCT GCC CAG AA - #G ACA GAT ACA TCC CAC          150                                                                          Ala Glu Asp Pro Gln Gly Asp Ala Ala Gln Ly - #s Thr Asp Thr Ser His           #     35                                                                      - CAT GAT CAG GAT CAC CCA ACC TTC AAC AAG AT - #C ACC CCC AAC CTG GCT          198                                                                          His Asp Gln Asp His Pro Thr Phe Asn Lys Il - #e Thr Pro Asn Leu Ala           # 55                                                                          - GAG TTC GCC TTC AGC CTA TAC CGC CAG CTG GC - #A CAC CAG TCC AAC AGC          246                                                                          Glu Phe Ala Phe Ser Leu Tyr Arg Gln Leu Al - #a His Gln Ser Asn Ser           #                 70                                                          - ACC AAT ATC TTC TTC TCC CCA GTG AGC ATC GC - #T ACA GCC TTT GCA ATG          294                                                                          Thr Asn Ile Phe Phe Ser Pro Val Ser Ile Al - #a Thr Ala Phe Ala Met           #             85                                                              - CTC TCC CTG GGG ACC AAG GCT GAC ACT CAC GA - #T GAA ATC CTG GAG GGC          342                                                                          Leu Ser Leu Gly Thr Lys Ala Asp Thr His As - #p Glu Ile Leu Glu Gly           #        100                                                                  - CTG AAT TTC AAC CTC ACG GAG ATT CCG GAG CC - #T CAG ATC CAT GAA GGC          390                                                                          Leu Asn Phe Asn Leu Thr Glu Ile Pro Glu Pr - #o Gln Ile His Glu Gly           #   115                                                                       - TTC CAG GAA CTC CTC CGT ACC CTC AAC CAG CT - #C CAG CTG ACC ACC GGC          438                                                                          Phe Gln Glu Leu Leu Arg Thr Leu Asn Gln Le - #u Gln Leu Thr Thr Gly           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - AAT GGC CTG TTC CTC AGC GAG GGC CTG AAG CT - #A GTG GAT AAG TTT TTG          486                                                                          Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Le - #u Val Asp Lys Phe Leu           #               150                                                           - GAG GAT GTT AAA AAG TTG TAC CAC TCA GAA GC - #C TTC ACT GTC AAC TTC          534                                                                          Glu Asp Val Lys Lys Leu Tyr His Ser Glu Al - #a Phe Thr Val Asn Phe           #           165                                                               - GGG GAC ACC GAA GAG GCC AAG AAA CAG ATC AA - #C GAT TAC GTG GAG AAG          582                                                                          Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile As - #n Asp Tyr Val Glu Lys           #       180                                                                   - GGT ACT CAA GGG AAA ATT GTG GAT TTG GTC AA - #G GAG CTT GAC AGA GAC          630                                                                          Gly Thr Gln Gly Lys Ile Val Asp Leu Val Ly - #s Glu Leu Asp Arg Asp           #   195                                                                       - ACA GTT TTT GCT CTG GTG AAT TAC ATC TTC TT - #T AAA GGC AAA TGG GAG          678                                                                          Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Ph - #e Lys Gly Lys Trp Glu           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - AGA CCC TTT GAA GTC AAG GAC ACC GAG GAA GA - #G GAC TTC CAC GTG GAC          726                                                                          Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Gl - #u Asp Phe His Val Asp           #               230                                                           - CAG GTG ACC ACC GTG AAG GTG CCT ATG ATG AA - #G CGT TTA GGC ATG TTT          774                                                                          Gln Val Thr Thr Val Lys Val Pro Met Met Ly - #s Arg Leu Gly Met Phe           #           245                                                               - AAC ATC CAG CAC TGT AAG AAG CTG TCC AGC TG - #G GTG CTG CTG ATG AAA          822                                                                          Asn Ile Gln His Cys Lys Lys Leu Ser Ser Tr - #p Val Leu Leu Met Lys           #       260                                                                   - TAC CTG GGC AAT GCC ACC GCC ATG TTC TTC CT - #G CCT GAT GAG GGG AAA          870                                                                          Tyr Leu Gly Asn Ala Thr Ala Met Phe Phe Le - #u Pro Asp Glu Gly Lys           #   275                                                                       - CTA CAG CAC CTG GAA AAT GAA CTC ACC CAC GA - #T ATC ATC ACC AAG TTC          918                                                                          Leu Gln His Leu Glu Asn Glu Leu Thr His As - #p Ile Ile Thr Lys Phe           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - CTG GAA AAT GAA GAC AGA AGG TCT GCC AGC TT - #A CAT TTA CCC AAA CTG          966                                                                          Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Le - #u His Leu Pro Lys Leu           #               310                                                           - TCC ATT ACT GGA ACC TAT GAT CTG AAG AGC GT - #C CTG GGT CAA CTG GGC         1014                                                                          Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Va - #l Leu Gly Gln Leu Gly           #           325                                                               - ATC ACT AAG GTC TTC AGC AAT GGG GCT GAC CT - #C TCC GGG GTC ACA GAG         1062                                                                          Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Le - #u Ser Gly Val Thr Glu           #       340                                                                   - GAG GCA CCC CTG AAG CTC TCC AAG GCC GTG CA - #T AAG GCT GTG CTG ACC         1110                                                                          Glu Ala Pro Leu Lys Leu Ser Lys Ala Val Hi - #s Lys Ala Val Leu Thr           #   355                                                                       - ATC GAC GAG AAA GGG ACT GAA GCT GCT GGG GC - #C ATG TTT TTA GAG CGC         1158                                                                          Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Al - #a Met Phe Leu Glu Arg           360                 3 - #65                 3 - #70                 3 -       #75                                                                           - ATA CCC AGG TCT ATC CCC CCC GAG GTC AAG TT - #C AAC AAA CCC TTT GTC         1206                                                                          Ile Pro Arg Ser Ile Pro Pro Glu Val Lys Ph - #e Asn Lys Pro Phe Val           #               390                                                           - TTC TTA ATG ATT GAA CAA AAT ACC AAG TCT CC - #C CTC TTC ATG GGA AAA         1254                                                                          Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pr - #o Leu Phe Met Gly Lys           #           405                                                               - GTG GTG AAT CCC ACC CAA AAA TAACTGCTCG CTCCTCAAC - #C CCTCCCCTCC            1305                                                                          Val Val Asn Pro Thr Gln Lys                                                           410                                                                   #           1356TCCCTGG ATGACATTAA AGAAGGGTTG AGCTGGAAAA A                    - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 414 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Met Pro Ser Ser Val Ser Trp Gly Ile Leu Le - #u Leu Ala Gly Leu Cys         #                 15                                                          - Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pr - #o Gln Gly Asp Ala Ala         #             30                                                              - Gln Lys Thr Asp Thr Ser His His Asp Gln As - #p His Pro Thr Phe Asn         #         45                                                                  - Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Ph - #e Ser Leu Tyr Arg Gln         #     60                                                                      - Leu Ala His Gln Ser Asn Ser Thr Asn Ile Ph - #e Phe Ser Pro Val Ser         # 80                                                                          - Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gl - #y Thr Lys Ala Asp Thr         #                 95                                                          - His Asp Glu Ile Leu Glu Gly Leu Asn Phe As - #n Leu Thr Glu Ile Pro         #           110                                                               - Glu Pro Gln Ile His Glu Gly Phe Gln Glu Le - #u Leu Arg Thr Leu Asn         #       125                                                                   - Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Ph - #e Leu Ser Glu Gly Leu         #   140                                                                       - Lys Leu Val Asp Lys Phe Leu Glu Asp Val Ly - #s Lys Leu Tyr His Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Gl - #u Glu Ala Lys Lys Gln         #               175                                                           - Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gl - #y Lys Ile Val Asp Leu         #           190                                                               - Val Lys Glu Leu Asp Arg Asp Thr Val Phe Al - #a Leu Val Asn Tyr Ile         #       205                                                                   - Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Gl - #u Val Lys Asp Thr Glu         #   220                                                                       - Glu Glu Asp Phe His Val Asp Gln Val Thr Th - #r Val Lys Val Pro Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Lys Arg Leu Gly Met Phe Asn Ile Gln Hi - #s Cys Lys Lys Leu Ser         #               255                                                           - Ser Trp Val Leu Leu Met Lys Tyr Leu Gly As - #n Ala Thr Ala Met Phe         #           270                                                               - Phe Leu Pro Asp Glu Gly Lys Leu Gln His Le - #u Glu Asn Glu Leu Thr         #       285                                                                   - His Asp Ile Ile Thr Lys Phe Leu Glu Asn Gl - #u Asp Arg Arg Ser Ala         #   300                                                                       - Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gl - #y Thr Tyr Asp Leu Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Va - #l Phe Ser Asn Gly Ala         #               335                                                           - Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Le - #u Lys Leu Ser Lys Ala         #           350                                                               - Val His Lys Ala Val Leu Thr Ile Asp Glu Ly - #s Gly Thr Glu Ala Ala         #       365                                                                   - Gly Ala Met Phe Leu Glu Arg Ile Pro Arg Se - #r Ile Pro Pro Glu Val         #   380                                                                       - Lys Phe Asn Lys Pro Phe Val Phe Leu Met Il - #e Glu Gln Asn Thr Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Pro Leu Phe Met Gly Lys Val Val Asn Pr - #o Thr Gln Lys                 #               410                                                           - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #site     (A) NAME/KEY: Modified                                                        (B) LOCATION: 1..4                                                  #/label=Modified siteNFORMATION:                                                             / note=- #"The amino terminus us derivatized by a                             butoxycarbon - #yl group, and the carboxyl terminus                           is deriva - #tized by a 4-methylcoumaryl-7-amide group."       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Arg Val Arg Arg                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #site     (A) NAME/KEY: Modified                                                        (B) LOCATION: 1..4                                                  #/label=Modified siteNFORMATION:                                                             / note=- #"The amino terminus us derivatized by a                             butoxycarbon - #yl group, and the carboxyl terminus                           is deriva - #tized by a para-nitroanilide group."              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Ala Ala Pro Ala                                                             __________________________________________________________________________

What we claim is:
 1. A furin endoprotease inhibitor comprising afragment of an α₁ -antitrypsin variant having an amino acid sequencecomprising the amino acids -Arg-Xaa-Xaa-Arg-, wherein Xaa is any aminoacid, at positions 355-358 of the native α₁ -antitrypsin amino acidsequence (SEQ ID No.: 6).
 2. The furin endoprotease inhibitor of claim 1comprising the amino acid sequence -Arg-Ile-Pro-Arg- (SEQ ID No.: 10).3. A pharmaceutical composition comprising a therapeutically effectiveamount of a furin endoprotease inhibitor of claim 1 and apharmaceutically acceptable carrier or diluent.
 4. A homogenouscomposition of matter comprising a peptide fragment of α₁ -antitrypsinPortland (SEQ ID No.: 9) having an amino acid sequence comprising theamino acids -Arg-Xaa-Xaa-Arg-, wherein Xaa is any amino acid, atpositions 355-358 of the native α₁ -antitrypsin amino acid sequence (SEQID No.: 6) and produced by a cell culture transformed with therecombinant expression construct comprising a nucleic acid encoding thefragment of α₁ -antitrypsin Portland that expresses the fragment of α₁-antitrypsin Portland thereby.
 5. A peptide having an amino acidsequence of 4 to 20 amino acids comprising the amino acid sequence-Arg-Xaa-Xaa-Arg-, wherein each Xaa is any amino acid.
 6. A method ofinhibiting viral infection of cells comprising contacting the cells witha peptide according to claim
 5. 7. The method of claim 6 wherein thevirus is cytomegalovirus.
 8. A method of inhibiting bacterial infectionof cells comprising contacting the cells with a peptide according toclaim
 5. 9. The method of claim 8 wherein the bacterial toxin isdiphtheria toxin of Corynebacterium diptheriae.
 10. The method of claim8 wherein the bacterial toxin is anthrax toxin of Bacillus anthracis.11. The method of claim 8 wherein the bacterial toxin is Pseudomonasaerugenosa exotoxin.
 12. A method of blocking endoproteolytic activationof a bacterial toxin comprising the step of contacting a cell in thepresence of the toxin with a furin endoprotease inhibitor comprising anα₁ -antitrypsin variant or peptide fragment thereof having an amino acidsequence comprising the amino acids -Arg-Xaa-Xaa-Arg-, wherein Xaa isany amino acid, at positions 355-358 of the native α₁ -antitrypsin aminoacid sequence (SEQ ID No.: 6).
 13. The method of claim 12 wherein thebacterial toxin is diphtheria toxin of Cornyebacterium diptheriae. 14.The method of claim 12 wherein the bacterial toxin is anthrax toxin ofBacillus anthracis.
 15. The method of claim 12 wherein the bacterialtoxin is Pseudomonas aerugenosa exotoxin.
 16. A method of inhibitingbacterial infection of cells comprising contacting the cells with afurin endoprotease inhibitor comprising an α₁ -antitrypsin variant orpeptide fragment thereof having an amino acid sequence comprising theamino acids -Arg-Xaa-Xaa-Arg-, wherein Xaa is any amino acid, atpositions 355-358 of the native α₁ -antitrypsin amino acid sequence (SEQID NO.: 6).
 17. A method of inhibiting viral infection of cellscomprising contacting the cells with a furin endoprotease inhibitorcomprising an α₁ -antitrypsin variant or peptide fragment thereof havingan amino acid sequence comprising the amino acids -Arg-Xaa-Xaa-Arg-,wherein Xaa is any amino acid, at positions 355-358 of the native α₁-antitrypsin amino acid sequence (SEQ ID No.: 6).
 18. The method ofclaim 17 wherein the virus is cytomegalovirus.